1. Field of the Invention
The invention relates to novel methods employing immobilized enzyme biosensors to determine micromolar levels of lead. Also disclosed are methods of releasing lead from complexes to make the lead available for detection, by conventional assay or by various electrochemical methods based on enzyme catalyzed reactions. The disclosed methods are adaptable to the rapid, efficient detection of lead ion in whole blood, employing a unique metal replacement of lead ion from a capture complex.
2. Description of Related Art
Heavy metals such as lead have received increasing recognition as serious threats to the environment and to human health. The effects of lead may not be acute; indeed chronic toxicity is of particular concern because this metal accumulates in tissues over a period of long-term exposure. This may lead to mental and physical abnormalities, especially in the very young.
The detrimental effects of lead in the environment have long been recognized. Lead poisoning has been detected in waterfowl due to lead shot. The elimination of tetraethyl lead as an octane booster in gasoline was part of an effort to prevent this metal from further contamination of soil and water supplies. However, the use of lead in glazes, paints and coatings, to mention a few examples, has occurred over long periods of time; in fact, lead in pottery may have contributed to the demise of earlier civilizations.
As a result of past long-term use of lead in a wide range of products, it is difficult to avoid exposure to this element. Lead solder joints in water pipes, for example, contribute to the lead content of drinking water. Modern interior paints are lead-free, but in older homes there may be significant exposure to lead in the surroundings from older lead-based paints, even when such paint layers are coated with the newer lead-free paints. Unfortunately, this has created a real risk of lead toxicity for those groups most susceptible, especially children.
The long term effect of lead on the health of children exposed to unacceptable levels is calculated to be very significant. This will ultimately reflect in higher health costs, due to increased disability and treatment required. This is of concern to health care professionals and to the federal government, to the extent that new rules related to a "threshold of concern" have been provided in guidelines set by Health & Human Services' Centers for Disease Control (C&E News, 1991). It is hoped that programs being developed to detect the presence of lead in groups at risk for the most damage from lead poisoning will lead to rapid, reliable methods of detecting low levels of lead in individuals. Unfortunately, it is difficult at best to detect lead in body fluids such as blood and it would be impractical to take tissue samples, for example brain tissue samples, to determine lead concentrations.
A simple, reliable method of detecting levels of lead in blood is not available. Current technology relies on time-consuming methods such as computerized stripping potentiometry (Almestrand, et al., 1988). Although the instrumentation required for this determination is not unduly complex, skilled technicians are needed.
Instruments currently available for monitoring trace metals generally require highly trained personnel to perform relatively sophisticated techniques. Consequently, analyses are performed in centralized laboratories set up for routine multiple sample analysis. However, there is no instrumentation available for use in the field or in the physician's office allowing rapid metal determinations with simple portable instruments that do not require highly technically trained personnel.
Trace heavy metal determination based on metal-enzyme interaction has taken advantage of either activation or inhibition of an enzyme by a metal, usually specific for the enzyme. Fluoride has been measured by its inhibition of liver esterase catalysis of a butyrate substrate (Linde, 1959) and magnesium has been measured in plasma by isocitric dehydrogenase activation (Baum and Czok, 1959). Titration determinations or rates of TPNH formation measured spectrophotometrically have been reported to be useful for measuring levels of activating metals such as manganese, magnesium and cobalt. Inhibiting metals such as lead can also be measured (Kratochvil, et al., 1967).
Analysis of lead based on inhibition of an enzyme's ability to produce hydrogen peroxide and oxidize homovanillic acid to a fluorescent product has also been explored. Horseradish peroxidase inhibition was linear over a range of 10-185 .mu.g/ml of lead (Guilbault, et al., 1968). Metal ion inhibition of the enzyme glucose oxidase with mercury(II), Ag(I) and Pb(II) has suggested that these metals are detectable at low levels although strong buffer-interactions were obtained when lead was present, casting doubt on the viability of the method generally to measure lead in trace amounts (Toren and Burger, 1968).
Of a few reported enzyme-inhibitor electrodes employed to measure trace heavy metals, most use CO.sub.2 and pH electrodes (Tran-Minh et al., 1990; Botre et al., 1983) which have a small, nonlinear response. Potentiometric sensors typically are used to detect the enzymatic reaction product, not the enzyme activity directly. The response time to inhibitors tends to be long because the inhibition manifests only after the product has diffused away from the electrode surface. When a pH electrode is used, the signal largely depends on the pH and the buffer capacity of the sample solution.
While an enzyme inhibitor sensor is desirable for selective measurement of lead ion, additional problems are encountered, particularly in whole blood samples where blood components, particularly blood proteins, tend to interfere with lead/enzyme interactions. Methods to determine lead levels directly in whole blood samples in the presence of interferants are desirable, and particularly one would seek to employ enzyme sensors which are particularly sensitive to inhibition by lead ion. The challenge therefore is to develop a rapid, selective method of detecting lead in blood without excessive manipulation, complex equipment or the need for highly skilled personnel.